Protein phosphorylation is utilized universally as a mechanism of signal transduction. However, the use of tyrosine phosphorylation by bacteria has been a matter of dispute. Conventional wisdom dictated that "prokaryotic phosphorylation" was typified by phosphorylation of histidine and aspartate residues of proteins, while "eukaryotic phosphorylation" was characterized by modification of serine, threonine, or tyrosine residues. Increasing numbers of reports have emerged challenging the traditional view of "prokaryotic" and "eukaryotic" phosphorlyation. One of the strongest links unifying prokaryotic and eukaryotic protein phosphorylation to date is IphP, a genomically-encoded dual-specificity protein phosphatase from the cyanobacterium Nostoc commune UTEX 584 bearing the active-site signature sequence of eukaryotic tyrosine-specific and dual-specificity protein phosphatases.

The catalytic properties and substrate specificity of IphP were examined in detail. The enzyme was able to discriminate among a variety of exogenous peptides and proteins. Kinetic studies revealed that IphP favors protein / peptide substrates over low molecular weight compounds.

Heparin effected IphP activity in a substrate-dependent manner. Enzyme activity toward casein (P-Ser) and MAP kinase (P-Thr/P-Tyr) was stimulated in the presence of the polyanion, wheras activity was inhibited by heparin toward other protein substrates. Both stimulation and inhibition by heparin were dose-dependent. The ability to stimulate IphP activity toward select substrates was attributed to the ability of heparin to recruit the enzyme and substrate to the same microenvironment.

To facilitate future genetic studies examining the role of tyrosine phosphorylation in cyanobacteria, we searched for evidence of protein tyrosine phosphorylation in Anabaena PCC 7120. In a collaborative effort with the laboratory of Dr. Potts, tyrosine phosphorylated proteins were identified in Anabaena utilizing several approaches, including comparative labelling with alpha- vs gamma-32P-ATP, phosphoamino acid analysis, and selective hydrolysis with a tyrosine specific protein phosphatase. Together, these data unequivocally demonstrate the presence of tyrosine-phosphorylated proteins in Anabaena PCC 7120.

Extracts of Anabaena PCC 7120 were examined for protein tyrosine phosphatase activity. An apparent PTP activity was detected, partially purified, and characterized. The protein phosphatase was ~38kDa by SDS-PAGE and sucrose density gradient centrifugation and displayed dual-specificity protein phosphatase (DSP) activity in vitro. The enzyme was localized to the periplasm and was thus assigned the title PAD, for Periplasmic Anabaena DSP. Periplasmic phosphoproteins of ~120 and 55 kDa that had been radiolabelled in vitro were dephosphorylated by partially purified PAD. PAD activity varied in vivo ~5-fold in a rhthymic, seemingly diurnal manner. Periplasmic proteins, including the 55kDa protein, were labelled in vivo and the degree of radiolabel incorporated into these proteins varied inversely with PAD activity.